Spatially-ordered multi-tissue structures such as the tendon-bone junction (TBJ) present unique, unmet challenges for the field of tissue engineering. The TBJ contains a graded interfacial zone linking tendinous and osseous compartments that is critical for its mechanical competence but is also a common injury site. Re-tears are frequent after clinical intervention because current surgical strategies forsake interface regeneration (biological fixation) for mechanical fixation of tendon to bone. The graded architectural and biochemical cues critical for the morphogenesis, remodeling, and function of multi-tissue structures such as the TBJ suggest that recapitulating their spatially-ordered design may be of significant functional importance for regenerative medicine applications. With the goal of generating a single composite biomaterial capable of guiding enthesis regeneration, we propose to explore how bioinspired structural and growth factors cues can induce spatially- selective, multi-lineage mesenchymal stem cell (MSC) differentiation using a unique collagen-GAG (CG) scaffold platform. We hypothesize that overlapping patterns of biomaterial structural (anisotropy, mineral content) and biochemical (tethered growth factors) signals are required for functional regeneration of the continuous anatomic insertion between tendon and bone. The objective of this proposal is therefore to investigate mechanisms by which spatially-graded structural and biochemical signals can synergistically induce multi-lineage MSC differentiation. We will leverage a prototype TBJ scaffold containing distinct tendinous (non- mineralized, anisotropic) and osseous (mineralized) compartments to investigate mechanisms by which MSCs integrate physical (scaffold anisotropy, mineral content) and biochemical (GDF-5, BMP-2) cues to generate a coordinated response to a series of unique cellular microenvironments. Aim 1 will define the combined influence of scaffold structural and biomolecular cues on autocatalytic induction of enthesis-specific differentiation profiles. Here, we will examine whether distinct scaffold compartments induce synergistic activation of mechanotransduction and TGF- superfamily signaling pathways in order to bias multi-lineage hMSC differentiation profiles. Aim 2 will examine the contribution of spatially-organized microstructural and biomolecular cues on regionally-distinct neo-TBJ tissue formation. We will assess formation of neo-enthesis tissue structures using both an in vitro bioreactor and an in vivo murine osteotendinous regeneration model. This advanced reductionist approach allows us to replicate potential regulatory elements of the native TBJ while retaining the ability to manipulate biomaterial structural and biomolecular cues in defined increments. Such information will be critical for designing biomaterial-based rheostats to control multiple hMSC fate decisions in close spatial register. Ultimately this approach may identify scaffold variants able to be seeded with autologous hMSCs immediately prior to implantation in order to regenerate a stable tendon-bone enthesis.